Detection of Reticulocytes, RNA and DNA

ABSTRACT

Reticulocytes, RNA or DNA are stained with a dye for detection in a flow cytometer. The dye has the formula: ##STR1## Wherein X=O, S, se or C(CH 3 ) 2  ; 
     R 1  =alkyl having from 1-6 carbons; 
     R 2  =alkyl having from 1-6 carbons; 
     R 3  =fused benzene, alkyl (having 1-6 carbons), methoxy or is absent; 
     R 4  =alkyl having 1-6 carbons, methoxy or is absent; and 
     n=zero or an integer from 1-6.

This is a division of Ser. No. 08,097, now U.S. Pat. No. 4,883,867,issued Nov. 28, 1989, which is a continuation-in-part of U.S. patentapplication Ser. No. 793,813, filed on Nov. 1, 1985, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to the detection and enumerationof reticulocytes in a blood sample. More particularly, the presentinvention relates to a dye which is suitable for staining ribonucleicacid polymers (RNA) and deoxyribonucleic acid (DNA) and is particularlysuitable for staining reticulocytes. The invention further relates to afluorescent composition.

2. Description of the Prior Art

In many cases, there is a need to detect RNA or RNA containingsubstances. Thus, for example, reticulocytes are a substance known tocontain RNA, and detection and enumeration of reticulocytes in a bloodsample are of value to clinicians. The reticulocyte count of a bloodsample is used as an indicator of erythropoietic activity, hasdiagnostic and prognostic value in acute hemorrhage and hemolyticanemia, and is a measure of response to iron, vitamin B₁₂ and folic acidtherapy. As known in the art, reticulocytes are precursors to mature redblood cells, and hence the term reticulocyte embraces the evolution anddevelopment of the cell whereby a mature red blood cell is generated.

In the past, reticulocytes in a blood sample have been determined byboth manual and automated methods by using appropriate stains such asnew methylene blue (NMB), brilliant cresyl blue (BCB), acridine orangeand pyronin Y.

Vital staining with the dye new methylene blue is considered to be thereference method for reticulocyte determinations, and in use this dyeprecipitates RNA. The method is manual, requires counting large numbers(for example, 500 to 1,000) of cells with a microscope, is slow,tedious, and subject to errors New methylene blue is nonfluorescent andtrue precipitated RNA is often difficult to differentiate fromprecipitated stain.

Acridine orange has had some use in staining reticulocytes by bothmanual and automated procedures. Acridine orange also precipitates RNA,and this prevents quantitative estimates of RNA content because ofpotential quenching. Moreover, acridine orange does not lead to adiffuse fluorescent distribution of stained cells. Age profiles of thecells (based on RNA content being proportional to fluorescence) are notreliable. Acridine orange has a great affinity for the plastic tubing inflow cytometers which leads to increased background and lengthyprocedures for removing the dye from the flow cytometer tubing. Inaddition, acridine orange stained cells are difficult to separate fromthe autofluorescent red cell peak, and the reticulocyte count is usuallylower than that obtained with new methylene blue.

The use of pyronin Y requires prior fixation of the erythrocytes withformalin, is cumbersome, time consuming, and generally yields poorresults. Moreover, pyronin Y has a very low quantum efficiency, leadingto very low fluorescent signals.

U.S. patent application Ser. No. 460,144 filed Jan. 24, 1983, relates toa method for detecting reticulocytes utilizing thioflavin T as a dye forstaining reticulocytes.

A dye for staining reticulocytes preferably has the followingproperties:

1. The dye should not fluoresce in the absence of RNA.

2. The dye should have a good fluorescent quantum yield.

3. The dye must be able to penetrate the membrane of cells containingRNA.

4. The dye should preferably have an excitation peak at about 488 nm.

None of the aforementioned known dyes for RNA and reticulocytes have allof the desirable features described hereinabove.

Accordingly, there is a need for providing a dye better suited forstaining reticulocytes so as to provide a procedure for accuratelydetermining reticulocytes in a blood sample.

In accordance with one aspect of the present invention, there isprovided an improvement in a process for detecting reticulocytes whereinthe reticulocytes are stained with a dye having the following formula:##STR2## Wherein

X═O, S, Se or C(CH₃)₂ ;

R₁ =alkyl having from 1-6 carbons;

R₂ =alkyl having from 1-6 carbons;

R₃ =fused benzene, alkyl (having 1-6 carbons), methoxy or is hydrogen;

R₄ =alkyl having 1-6 carbons, methoxy or is hydrogen; and n=zero or aninteger from 1-6

In accordance with another aspect of the present invention,reticulocytes are detected in a flow cytometer after the reticulocyteshave been stained with the dye of the invention.

In accordance with a further aspect of the present invention, there isprovided a composition comprised of reticulocytes stained with the dyeof the invention. The dye of the invention differs structually fromthioflavin T. Thioflavin T has the following structure: ##STR3## Forconvenience in the description hereinbelow and to describe a preferredembodiment of the invention, the dye of the invention for stainingreticulocytes where R₁ ═R₂ ═CH₃ ; R₃ ═R₄ ═H, X═S and n═0 is referred toas "thiazole orange".

Applicant has found that thiazole orange is an effective dye forstaining reticulocytes. The use of thiazole orange offers the furtheradvantage that thiazole orange when unbound to ribonucleic acid provideslittle or no fluorescence, whereas thiazole orange when bound toribonucleic acid in the reticulocytes is fluorescent. Thiazole orangecan be excited at 488 nm whereas thioflavin T is excited at a maximum ofabout 455 nm.

In accordance with the present invention, when staining reticulocytes ina blood sample, the dyes of the invention may be employed as an aqueoussolution, and in particular as an isotonic saline solution, which maycontain a minor amount of methanol. The blood sample, which may be wholeblood or a blood fraction, is stained with the dye by mixing the bloodsample with the solution of thiazole orange. It has been found that byusing thiazole orange as the stain, it is possible to detect andenumerate reticulocytes in a whole blood sample.

The dyes of the invention exhibit a strong absorption peak (unbound) inthe range of from about 470 nm to about 600 nm; however, in the unboundstate, the dye does not provide either a detectable excitation oremission peak. When thiazole orange stains the RNA in the reticulocytes,the optical properties thereof change dramatically. In particular, theabsorption curve shifts to a longer wavelength, and the dye now exhibitsstrong fluorescence. For thiazole orange, the excitation maximum is atabout 510 nm, and the emission maximum is at about 530 nm, giving aStokes shift of about 20 nm. As a result of the excitation peak of thebound dye being in the order of about 510 nm, in using the automaticflow cytometer, the light source may be a mercury lamp which has anenergy line at about 485 nm or an argon ion laser which has strongemission at 488 nm. The lack of fluorescence of the dye when not boundto nucleic acid provides low backgrounds and allows an operator toselect a fluorescent threshold (or "gates") for an automatic flowcytometer by simply running an unstained control. Although excitationmay be effected at other wavelengths, the thiazole orange stainedreticulocytes are preferably excited at a wavelength of from about 460nm to about 520 nm.

The dyes of the invention do not precipitate RNA, and as a result, thestained reticulocyte cells maintain a relatively homogeneousdistribution of intracellular RNA, whereby there is a nearly linearrelationship between the fluorescent signal measured for an individualreticulocyte and its RNA content Clinically, this provides the physicianwith additional information beyond the reticulocyte count in that RNAcontent is a function of reticulocyte age. Accordingly, by using a dyeof the invention, a clinician has the ability to do reticulocyte ageprofiles as well as simple reticulocyte counts.

The use of dyes of the invention for staining reticulocytes in a bloodsample offers the further advantage that the fluorescent signals fromthe stained reticulocytes are well separated from those of the matureerythrocytes, whereby results can be directly read in an automatic lowcytometer without extensive data manipulation.

Reticulocytes, RNA or DNA stained with a dye of the invention, althoughpreferably enumerated in an automatic flow cytometer, can also becounted by a manual procedure or automated microscopy.

Automatic flow cytometers are well known in the art, and the presentinvention is not limited to the use of any particular flow cytometer.Thus, for example, thiazole orange stained reticulocytes may be detectedand enumerated in the FACS 440™ flow cytometer or the FACS Analyzer™flow cytometer, both sold by Becton Dickinson and Company. In using suchautomatic flow cytometers, fluorescent gates are set by use of anunstained control sample, and the fluorescent gates are then used on thestained sample.

The use of an automatic flow cytometer for detection and enumeration ofreticulocytes stained with thiazole orange provides results whichclosely correlate with results obtained by a known standard method forenumerating reticulocytes which uses methylene blue or acridine orange.

The use of reticulocytes stained with thiazole orange in an automaticflow cytometer is particularly advantageous in that there are lowfluorescent backgrounds and fluorescent gates may be easily selected byuse of an unstained control. Moreover, there is no precipitation ofintracellular reticulocyte RNA, whereby the cells need not be fixed. Inaddition, there is a linear relationship between the fluorescent signalfor an individual reticulocyte, which provides information as toreticulocyte age.

Still another advantage of the present invention is that thiazole orangestained reticulocytes can be used in an automatic flow cytometer havinglower sensitivities, e.g., one may use a mercury arc lamp as opposed toan argon laser.

Although Flow Cytometry and Sorting, pages 457-58 Edited by Melamed etal., John Wiley & Sons, describes the use of both acridine orange andthioflavin T for staining RNA of living cells, this publication does notdisclose the use of the dyes of the invention as a stain forreticulocyte detection and enumeration in an automatic flow cytometer.

The following example illustrates various features of the presentinvention but is not intended to in any way limit the scope of theinvention as set forth in the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 (1A and 1B) shows a fluorescence vs. forward scatter, andfluorescence histogram for normal, unstained, and thiazole orange,respectively.

FIG. 2 (2A and 2B) shows a fluorescence vs. forward scatter, andfluorescence histogram for normal blood stained with thiazole orange,respectively.

FIG. 3 (3A and 3B) shows a fluorescence vs. forward scatter, andfluorescence histogram for unstained anemic blood (17.3% reticulocytesby new methylene blue assay), respectively.

FIG. 4 (4A and 3B) shows a fluorescence vs. forward scatter, andfluorescence histogram for anemic blood after staining, respectively.

FIG. 5 (5A and 5B and 5C) shows a fluorescence vs. forward scatter, andfluorescence histogram for anemic blood (8.4% reticulocytes by newmethylene blue assay) stained with thiazole orange for varying lengthsof time: 30 minutes: 70 minutes: 7 hours, respectively.

FIG. 6 shows a comparison of manual analysis with new methylene blue andflow cytometry analysis using thiazole orange.

FIG. 7 shows fluorescence emission spectra of thiazole orange with DNA.RNA phosphate buffered saline (PBS) which was free of nucleic acids.

EXAMPLE 1

A dye of the invention wherein R₁ and R₂ ═CH₃, X═S n═0 and R₃ and R₄ arehydrogen (thiazole orange) was used in a procedure for reticulocytestaining.

A 1 mg/ml stock solution of the dye in methanol was prepared. A 1:8,000dilution of the stock solution in pH 7.2 phosphate buffered saline wasmade, 5 μL of whole blood was mixed into 1 mL of the diluted dyesolution. The sample was analyzed on a FACS 440™ flow cytometer with anexcitation wavelength of 488 nm and a 530/30 nm bandpass filter.

FIGS. 1-6 show FACS data for reticulocyte analysis of normal and anemicblood using thiazole orange of this example.

A solution of thiazole orange in DMSO was prepared (5×10⁻³ M). Thesolution was diluted into PBS (5×10⁻⁵ M). A cuvette containing 30 μL ofthe thiazole orange solution, 90 μL of DNA solution (1 mg/mL) and 2.88mL of PBS was mixed and the fluorescence emission spectrum measured on aPerkin-Elmer MPF-2A fluorescence spectrophotometer. The excitationwavelength was 500 nm and the emission was measured from 510 to 610 nm.A cuvette containing 30 μL of the thiazole orange solution, 50 μL of RNA(1 mg/mL) and 2.92 mL PBS was mixed and the fluorescence emissionspectrum measured in the same way as described above A cuvettecontaining 30 μL of the thiazole orange solution and 2.97 mL PBS wasmixed and the fluorescence emission spectrum measured.

EXAMPLE 2

A dye wherein R₁ ═R₂ ═CH₃,R₃ ═fused benzene, R₄ ═H, X═S and n═0(thiazole red), was shown to be fluorescent only in the presence ofnucleic acid polymers. FIG. 8 shows fluorescence emission spectra ofthiazole red with DNA, RNA and in PBS. The spectra were obtained in thesame way as described in Example 1 except the excitation wavelength was520 nm and the emission spectra were measured from 530 to 630 nm. Thestructure of thiazole red is: ##STR4##

EXAMPLE 3

A dye wherein R₁ ═R₂ ═CH₃, R₃ ═R₄ ═H, X═0 and n═0 (methyl oxazoleyellow), was shown to be fluorescent only when bound to nucleic acidpolymers. FIG. 9 shows fluorescence emission spectra of methyl oxazoleyellow with DNA, RNA and in PBS. The spectra were obtained in the sameway as described in Example 1 except the excitation wavelength was 480nm and the emission spectra were measured from 490 to 590 nm. Thestructure of methyl oxazole yellow is: ##STR5##

EXAMPLE 4

A dye wherein R₁ ═R₂ ═CH₃, R₃ ═R₄ ═H, X═S and n═1 (thiazole blue), wasshown to be fluorescent only in the presence of nucleic acid polymersFIG. 10 shows fluorescence emission spectra of thiazole blue with DNA,RNA and in PBS. The spectra were obtained in the same way as describedin Example 1 except the excitation wavelength was 630 and the emissionspectra were measured from 640 to 740 nm.

EXAMPLE 5

A dye wherein R₁ ═R₂ ═CH₂ CO₂ H, R₃ ═R₄ ═H, X═S and n═0 (thiazole orangedicarboxylate), was shown to be fluorescent only when bound to nucleicacid polymers. FIG. 11 shows fluorescence emission spectra of thiazoleorange dicarboxylate with DNA, RNA and in PBS. The spectra were obtainedin the same way as described in Example 1.

EXAMPLE 6

A dye wherein R₁ ═R₂ ═CH₃, R₃ ═R₄ ═OCH₃, X═S and n═0 (dimethoxy thiazoleorange), was shown to be fluorescent only when bound to nucleic acidpolymers FIG. 12 shows fluorescence emission spectra of dimethoxythiazole orange with DNA, RNA and in PBS The spectra were obtained inthe same way as described in Example 1 except the excitation wavelengthwas 510 nm and the emission spectra were measured from 520 to 620 nm.The structure of dimethoxy thiazole orange is: ##STR6##

Numerous modifications and variations of the present invention arepossible in light of the above teachings, and, therefore, are within thescope of the appended claims. The invention may be practiced otherwisethan as particularly described.

What is claimed is:
 1. In a process for detecting reticulocytes, RNA orDNA in a sample, the improvement comprising:adding to said sample afluorescent dye having the formula ##STR7## wherein: X=0, S, Se or C(CH₃)₂ ; R₁ =alkyl having from 1-6 carbons; R₂ =alkyl having from 1-6carbons; R₃ =fused benzene, alkyl (having 1-6 carbons), methoxy orhydrogen; R₄ =alkyl having 1-6 carbons, methoxy or hydrogen; and n=zeroor an integer from 1-6; exciting said sample with light of excitationwavelength; and measuring fluorescence emitted from said sample.
 2. Theprocess of claim 1 wherein the sample is excited and fluorescence ismeasured by means of a flow cytometer.
 3. The process of claim 1 whereinthe sample is excited and fluorescence is measured by means offluorescence microscopy.
 4. The process of claim 2 wherein the samplesexcited in the flow cytometer with light from a mercury arc lamp.
 5. Theprocess of claim 2 wherein the sample is excited in the flow cytometerwith light from an argon laser.
 6. The process of claim 2 wherein thesample comprises whole blood.
 7. The process of claim 6 wherein thesample is not fixed with a cell fixative.
 8. The process of claim 1wherein R₁ ═CH₃, R₂ ═CH₃, R₃ ═hydrogen, R₄ ═hydrogen, X═S and n═1. 9.The process of claim 1 wherein R₁ ═CH₃, R₂ ═CH₃, R₃ ═fused benzene, R₄═hydrogen, X═S and n═0.
 10. The process of claim 1 wherein R₁ ═CH₃, R₂═CH₃, R₃ ═hydrogen, R₄ ═hydrogen, X═0 and n═zero.